Following detection of a microorganism in a patient sample, it is often desirable to determine to which antibiotics the microorganism is susceptible. There are now a number of bacterial species which increasingly exhibit resistance to one or more classes of antimicrobial agents, making it that much more important to perform susceptibility testing. Failure of a particular susceptibility test to accurately predict antimicrobial resistance in a patient's isolate could significantly impact patient care if an antibiotic is used to which the microorganism is not susceptible.
Different types of susceptibility tests can be used to test a microorganism. The following brief descriptions give details of some known susceptibility tests as well as some details that relate to the present invention.
One type of susceptibility test is the disk diffusion test, often referred to as the Kirby-Bauer test. This is a standardized test that involves inoculating (with 0.5 McFarland standardized suspension of a microbial isolate) a gel plate (e.g. a 150-mm Mueller-Hinton agar plate) and placing thereon one or more disks impregnated with fixed concentrations of antibiotics. After incubation (e.g. 18-24 hours at 35 degrees C), the diameter of zones of inhibition around the disks (if present) determine the sensitivity of the inoculated microorganism to the particular antimicrobial agent impregnated in each disk. Due to the standardization of the Kirby-Bauer method, results of this method are analyzed by comparing the diameter of the inhibition zone with information published by NCCLS (National Committee on Clinical Laboratory Standards) in Performance Standards for Antimicrobial Disk Susceptibility Testing, the subject matter of which is incorporated herein by reference. The results of this test are semi-quantitative in that there are three categories of susceptibility--namely resistant, intermediate and susceptible. As can be seen in FIG. 1, an agar plate 10 with inoculum has a plurality of disks 12 placed thereon, which disks are impregnated with antibiotics (of different types and/or concentrations). After incubation, zones of microbial growth inhibition 14 are formed. These zones 14 are interpreted to be resistant, intermediate or susceptible based on NCCLS criteria.
Another method of antimicrobial susceptibility testing is the antibiotic gradient method. This test utilizes an antibiotic gradient in a gel medium. Paper or plastic strips are impregnated with an antibiotic concentration gradient. A plurality of strips are placed on a Mueller-Hinton agar plate like spokes on a wheel, with the plate having been inoculated with the microorganism to be tested. After incubation, an antibiotic gradient is formed in the gel in an elliptical shape around each test strip (if the microorganism is susceptible to the antibiotic on the particular strip). The minimum concentration of the antimicrobial agent that prevents visible microorganism growth is the endpoint of the test (the minimum inhibitory concentration, or MIC). Put in other words, in disk diffusion testing, the MIC is the concentration at the edge of the inhibition zone (the growth/no growth boundary). In this case, the MIC is the point at which the elliptical growth inhibition area intersects the test strip. As can be seen in FIG. 2, agar plate 1 has a plurality of test strips 3 that are impregnated with an antibiotic gradient. Elliptical zones 5 are formed where microorganism growth is inhibited by the antibiotic agent in/on the test strip. Point 7 where the elliptical zone intersects the test strip is the MIC point.
A third type of susceptibility test is the broth microdilution test. In this type of test, dilutions of antibiotics (e.g. consecutive two-fold dilutions) are prepared. Often, at least ten concentrations of a drug are prepared in tubes or microwells. Each tube or well having the various concentrations of antibiotics is inoculated with a particular microorganism (a standardized suspension of test bacteria is added to each dilution to obtain a final concentration of 5.times.10.sup.5 CFU/ml). A growth control well and an uninoculated control well are included on each plate. After incubation (e.g. for 16-24 hours at 35 degrees C), the wells or tubes are examined manually or by machine for turbidity, haze and/or pellet. Indicators can be placed in the wells to facilitate the visualization of microbial growth. As with other tests, the minimum concentration of antimicrobial agent that prevents visible microbial growth is the MIC.
Commercial microdilution tests are typically performed on standard 96 well plates, each well holding approximately 100 to 200 microliters with commercially prepared antibiotic test panels. With 96 wells and 2 to 10 different dilutions for each antibiotic, numerous antibiotics can be tested on a single plate. A significant problem with such commercial microdilution systems is the inflexibility of the standard antibiotic test panels. The commercial plates are manufactured with various amounts of frozen, dried or lyophilized antimicrobial agents in the wells. This avoids the time consuming task of preparing the plates. However, due to the availability of many antibiotics (more than fifty in the United States), it is often problematic for a laboratory to find a standard commercial test panel which is ideal for that laboratory's needs. FIG. 3 is an illustration of a 96-well plate used in such a microdilution system.
A variation of the broth microdilution method is set forth in U.S. Pat. No. 5,501,959. This system uses microtiter plates with 168 wells, each containing a paper disk attached to the bottom of the well. The disks contain serial two-fold dilution concentrations of various antimicrobial agents, as well as a redox indicator. Up to 20 different antimicrobial agents can be tested on a plate. This use of paper disks simplifies the manufacture of the custom panels. However, higher costs are involved when a susceptibility test is custom made for a customer.
Current instruments that offer the highest degree of automation in susceptibility testing are typically based on automating the tasks performed in the manual broth microdilution method mentioned above. One such example is the instrument described in U.S. Pat. No. 4,448,534. This instrument uses multi-well plates that are pre-loaded with serial two-fold dilution concentrations of antimicrobial agents. Plates are inoculated manually and placed in the instrument, where they are incubated. At the appropriate times, the wells on the plate are read by a photometer/fluorometer to determine the results of the test. Another automated system is described in U.S. Pat. No. 3,957,583. This instrument uses small multi-chamber cards that are pre-loaded with serial two-fold dilution concentrations of antimicrobial agents. Cards are inoculated automatically, incubated, and monitored within the instrument. This instrument reads the chambers in the card periodically using a photometer. These kinetic measurements yield growth curves that allow the instrument to determine the results of the test. Though the aforementioned instruments perform testing in 4 to 8 hours, they may fail to detect induced resistance of the microorganism, which could result in an incorrect susceptibility report. Unfortunately, the degree of automation that is provided by instruments based on broth microdilution is not available for methods such as disk diffusion.